1. Field of the invention:
This invention relates to a method for obtaining an estimate of the time integral (defined below) of the concentration of a substance in a biological fluid.
More particularly, it relates to a method for obtaining, for assay, a single aliquot of material, in which the content of the substance of interest provides a direct measure of the time integral of the concentration of the freely-diffusable fraction of that substance in the biological fluid over a known time interval; and the invention also provides a device useful in the practice of this method.
2. Nature and significance of time-integrated measurement of biochemical substances:
Very often a primary goal in making measurements of substances in biological fluids is to provide information about some enduring state of the organism which produces those fluids, such as when biochemical measures are taken to provide information for the diagnosis of disease.
It often happens that the concentrations of these substances in the biological fluids are subject to temporal fluctuations, such that a series of samples taken from the biological fluids would reveal a distribution of concentration values. Generally it is not the concentration present at a given instant, but rather the extended summation of the fluctating concentrations over some time interval, which is of ultimate interest. This summation of instantaneous levels over a defined time interval, in mathematical terms, would be called the "time-integral".
The time-integral is preferred because it is more representative of the enduring state of the organism than are the temporally-fluctuating instantaneous values. The time integral, divided by the length of the time interval over which it was obtained, is equivalent to the true average concentration of the substance over that interval.
This advantage of the time-integral over instantaneous values is illustrated by Goldzieher et al. (Journal of Clinical Endocrinology and Metabolism 43:824 (1976)). These authorities estimated the 8-hour mean of blood plasma luteinizing hormone concentrations in normal men by a prior art method (described below), then calculated the probability that a single plasma sample would yield a value within plus or minus 20 percent of this mean.
They estimated this probability was only 30%, i.e., that a single plasma measurement was a very poor indicator of the enduring state of the organism, with regard to this important substance.
The practical diagnostic advantage of time-integrated measurement can be illustrated by another example from the medical literature. Zadik et al. (Journal of Clinical Endocrinology and Metabolism 51:1099, (1980)) compared the ability of three clinical tests to distinguish among the three classes, i.e., normal persons, patients with mild essential hypertension, and patients with Cushing's syndrome. Three tests were used for each of those three classes of patients: (1) a prior-art method (described below) of estimating the time integral of plasma cortisol, (2) measurement of urinary free cortisol, and (3) measurement of urinary 17-hydroxycorticosteroids. The latter two methods are recognized to be far superior to instantaneous plasma cortisol measurement, and are considered to be the standard measures in current medical practice.
However, the time-integrated plasma cortisol measurement was found to be clearly superior to the standard measures in discriminating among these groups. The authors nevertheless declined to recommend the time-integrated measurement as a new standard of practice, however, because the prior art heretofore available for that improved method is exceedingly complex.
3. Nature and significance of measurement of the "free" fraction of biochemical substances:
It often happens that compounds of biological interest (e.g., hormones, drugs, amino acids, etc.) exist in biological fluids in two states or fractions, i.e., those molecules which are in association with macromolecules (generally proteins) such that their movements and reactions are in some way limited by the macromolecules, and those which are free of such association.
The latter fraction is often called the "free" fraction, and the former the "bound" fraction; and in general, the proportion of the bound and free fractions is determined by the concentrations of the various biochemical components of the biological fluid, by temperature, and by other factors of a physical or chemical nature.
When the compounds of interest exist in both "bound" and "free" states, it is often observed that the biological effects of the compounds seem to be exerted only by the free fraction (see for example Lasnitzki and Frankin, Journal of Endocrinology 64:289, (1975)).
Indeed, it is found that the free fraction concentration of a hormone in a biological fluid is a much better indicator of the state of the organism than is the total concentration. Vermeulen et al. (Journal of Endocrinology and Metabolism 33:758, (1971)) present many cases illustrating the superiority of measurements of free testosterone concentration obtained by a prior art method (described below), over simple measurement of the total hormone concentration, again in the context of medical diagnosis.